BMC Complementary and
Alternative Medicine
BioMed Central
Open Access
Research article
Induction of cell cycle changes and modulation of
apoptogenic/anti-apoptotic and extracellular signaling regulatory
protein expression by water extracts of I'm-Yunity™ (PSP)
Tze-chen Hsieh, Peili Wu†, Spencer Park† and Joseph M Wu*
Address: Department of Biochemistry & Molecular Biology, New York Medical College, Valhalla, NY 10595, USA
Email: Tze-chen Hsieh - [email protected]; Peili Wu - [email protected]; Spencer Park - [email protected];
Joseph M Wu* - [email protected]
* Corresponding author †Equal contributors
Published: 11 September 2006
BMC Complementary and Alternative Medicine 2006, 6:30
doi:10.1186/1472-6882-6-30
Received: 13 April 2006
Accepted: 11 September 2006
This article is available from: http://www.biomedcentral.com/1472-6882/6/30
© 2006 Hsieh et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: I'm-Yunity™ (PSP) is a mushroom extract derived from deep-layer cultivated mycelia of
the patented Cov-1 strain of Coriolus versicolor (CV), which contains as its main bioactive ingredient a family
of polysaccharo-peptide with heterogeneous charge properties and molecular sizes. I'm-Yunity™ (PSP) is
used as a dietary supplement by cancer patients and by individuals diagnosed with various chronic diseases.
Laboratory studies have shown that I'm-Yunity™ (PSP) enhances immune functions and also modulates
cellular responses to external challenges. Recently, I'm-Yunity™ (PSP) was also reported to exert potent
anti-tumorigenic effects, evident by suppression of cell proliferation and induction of apoptosis in malignant
cells. We investigate the mechanisms by which I'm-Yunity™ (PSP) elicits these effects.
Methods: Human leukemia HL-60 and U-937 cells were incubated with increasing doses of aqueous
extracts of I'm-Yunity™ (PSP). Control and treated cells were harvested at various times and analyzed for
changes in: (1) cell proliferation and viability, (2) cell cycle phase transition, (3) induction of apoptosis, (4)
expression of cell cycle, apoptogenic/anti-apoptotic, and extracellular regulatory proteins.
Results: Aqueous extracts of I'm-Yunity™ (PSP) inhibited cell proliferation and induced apoptosis in HL60 and U-937 cells, accompanied by a cell type-dependent disruption of the G1/S and G2/M phases of cell
cycle progression. A more pronounced growth suppression was observed in treated HL-60 cells, which
was correlated with time- and dose-dependent down regulation of the retinoblastoma protein Rb,
diminution in the expression of anti-apoptotic proteins bcl-2 and survivin, increase in apoptogenic proteins
bax and cytochrome c, and cleavage of poly(ADP-ribose) polymerase (PARP) from its native 112-kDa form
to the 89-kDa truncated product. Moreover, I'm-Yunity™ (PSP)-treated HL-60 cells also showed a
substantial decrease in p65 and to a lesser degree p50 forms of transcription factor NF-κB, which was
accompanied by a reduction in the expression of cyclooxygenase 2 (COX2). I'm-Yunity™ (PSP) also
elicited an increase in STAT1 (signal transducer and activator of transcription) and correspondingly,
decrease in the expression of activated form of ERK (extracellular signal-regulated kinase).
Conclusion: Aqueous extracts of I'm-Yunity™ (PSP) induces cell cycle arrest and alterations in the
expression of apoptogenic/anti-apoptotic and extracellular signaling regulatory proteins in human leukemia
cells, the net result being suppression of proliferation and increase in apoptosis. These findings may
contribute to the reported clinical and overall health effects of I'm-Yunity™ (PSP).
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Background
Throughout history, mushroom and mushroom products
have always been revered as food delicacies and are also
held in high esteem for their overall health benefits in
many cultures, particularly the Orient [1-4]. In East Asian
societies, a variety of mushrooms are sold either fresh or
as dietary supplements. These products are frequently
consumed depending on season of the year as prophylactic measures for common ills and to improve the general
well-being of individuals [5]. The notable regard mushrooms are given for promoting wellness of the public at
large is perhaps in part attributed to the rather extensive
anecdotal and scientific evidence reporting their disease
preventive properties, focusing mostly on the potentiation of immune functions and regulation of biological
responses [3,4,6]. Beginning in the 1990s, however, it has
become increasingly clear that mushrooms, mushroom
extracts, and indeed plant/botanical polysaccharides in
general, have activities beyond that of the immune system, with suppression of tumorigenesis having the most
medical relevance and significance [7-10]. Thus, for
instance, polysaccharides with 6-branched 1,3-β glucan
structures isolated from the cultured fruit body of edible
mushroom Sparassis crispa reportedly show antitumor
activity when tested against Sarcoma 180 in the ICR strain
mice [11,12]. Antineoplastic activity has been demonstrated in polysaccharides isolated from Pleurotus tuberregium [13], and from fruit body of cultivated Agricus
blazei [14,15]. Several polysaccharide-peptide, and
polysaccharide-protein complexes with immunomodulatory and antitumor activities have been isolated and purified from mycelia cultures of Tricholoma Sp., an edible
mushroom native to Hong Kong [16-18]. Maitake, a
mushroom indigenous to northeastern Japan, is recognized as a rich source of polysaccharides with a widerange of biological and medicinal properties [19,20].
Most notably, gel-purified D-fraction from Maitake characterized as heterogeneous β-(1→6)-branched β-(1→3)linked alkali-soluble and acid-insoluble polysaccharides
[21], show bioactivities spanning the control of immune
response, suppression of tumor proliferation, induction
of apoptosis, inhibition of metastasis, and regulation of
angiogenesis [10,21,22]. Additionally, mushrooms
reportedly also contain antitumor proteins capable of
inducing apoptosis as well as cell cycle checkpoint arrest
in cultured malignant cells [23].
Dietary supplements derived from edible mushroom
known as Yunzhi, or Coriolus versicolor (Trametes versicolor,
Fr.) – known as one of six Zhi's recorded in the "Shen Non
Compendium Medica" some 2000 years ago – reportedly
also show a number of medicinal properties [2,24,25].
Structural and functional analyses of Yunzhi have benefited from the discovery of the patented Cov-1 strain of
Coriolus versicolor in 1984–1987 by Yang and coworkers,
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through an exhaustive screen of a large number of strains
of Yunzhi [1,4,26]. Subsequently, an innovative industrial
scale cultivation method using the mycelia of Cov-1 was
developed, which led to the serendipitous discovery, isolation and purification of a family of polysaccharo-peptide, denoted I'm-Yunity™ (PSP) [4,25,27].
I'm-Yunity™ (PSP) has demonstrated potent immunomodulatory and antitumor activities in tissue culture
studies, based principally on data using flow cytometry
[27-31]. In previous studies, we have observed that ethanol and water extracts of I'm-Yunity™ (PSP) exerted doseand time-dependent anti-proliferative effects in human
promyelocytic HL-60 leukemic cells. Flow cytometric
analyses showed that low doses of ethanol and water
extracts of I'm-Yunity™ (PSP) induced partial cell arrest in
the G1 phase, whereas high doses resulted in apoptotic cell
death. Moreover, water extracts were found to have significantly more pronounced anti-cellular effects than ethanol extracts [28]. In addition, water and ethanol extracts
of I'm-Yunity™ (PSP) markedly increased the secretion of
IL-1β and IL-6 with concomitant reduction of IL-8 in HL60 cells, while having no affect on growth and lymphokine expression in normal human lymphocytes [28].
The molecular details by which I'm-Yunity™ (PSP) exerts
its biological effects remain incompletely understood.
To further investigate the mechanism of action of I'mYunity™ (PSP), we studied the effects of water extracts of
I'm-Yunity™ (PSP) on growth, viability and cell cycle
traverse using human HL-60 and U-937 leukemia cells.
We found a similar degree of inhibition of cell growth and
induction of apoptosis, accompanied by differential targeting of G1/S and G2/M cell cycle phase transition,
respectively, in the HL-60 and U-937 cells. Furthermore,
in HL-60 cells, we showed that growth suppression by
water extracts of I'm-Yunity™ (PSP) was correlated with
time- and dose-dependent inhibition of retinoblastoma
protein Rb expression. Correspondingly, induction of
apoptosis in I'm-Yunity™ (PSP)-treated cells – evident by
cleavage of poly(ADP-ribose) polymerase (PARP) from its
native 112-kDa form to the 89-kDa truncated product –
was matched by a significant decrease in the expression of
anti-apoptotic proteins bcl-2 and survivin, and an
increase in apoptogenic proteins bax and cytochrome c.
Moreover, HL-60 cells treated with I'm-Yunity™ (PSP) also
showed a diminished p65 and p50 forms of transcription
factor NF-κB, which paralleled the reduced expression of
COX2. We further observed that suppression of HL-60 cell
growth was positively correlated with an increase in signal
transducer and activator family of transcription factors
STAT1, and conversely, with reduction in the expression
of activated form of ERK. We postulate that the observed
cell cycle and apoptogenic/anti-apoptotic and extracellular signaling regulatory protein expression changes result-
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M I
M I M I
Stacking
gel
kDa
97.4
66.2
45
31
21.5
A
B
C
Figure
Analysis
(PSP)
A),
reagent
Schiff's
by1
from
of
SDS-PAGE
reagent
constituents
MP Biomedicals
from
and of
staining
Sigma
water
(panel
(panel
with
extracts
Coomassie
C)
B), and
of I'm-Yunity™
silver
bluestaining
(panel
Analysis of constituents of water extracts of I'm-Yunity™
(PSP) by SDS-PAGE and staining with Coomassie blue (panel
A), Schiff's reagent from Sigma (panel B), and silver staining
reagent from MP Biomedicals (panel C). In each panel, lane
M corresponds to the staining pattern of protein markers,
with arrows pointing to the position of migration of different
molecular weight proteins. Correspondingly, lane I shows the
migration of samples of water extracts of I'm-Yunity™ (PSP)
after they were separated on 10% SDS-PAGE. The patterns
revealed by different stains support the interpretation that
I'm-Yunity™ (PSP) is a family of heterogeneous carbohydrate-containing polypeptides.
ing from treatment by water extracts of I'm-Yunity™ (PSP)
are mechanistically linked to its reported clinical
attributes and general health beneficial effects.
Methods
Source of I'm-Yunity™ (PSP)
I'm-Yunity™ (PSP) was supplied by ICM Holdings Ltd.
(Hong Kong, China), as a mushroom product produced
from deep-layer cultivated mycelia of Cov-1 according to
Good Manufacturing Practice (GMP) standards. Briefly,
following a 2.5–3 day batch fermentation culture [27], the
biomass containing mycelia was extracted with hot water
at 95°C for 4–5 h. The aqueous extract was differentially
precipitated with ethanol, and fractionated using a proprietary scheme for enriching and retaining bioactive
polysaccharo-peptides with molecular weights >40-kDa
http://www.biomedcentral.com/1472-6882/6/30
and an average polysaccharide:peptide ratio of 2:1 [28].
Additional quality control of I'm-Yunity™ (PSP) involved
determination of heavy metal contents, microorganism
contamination, authentication of elution profiles on high
pressure liquid chromatograph, and supplementary bioactivity-guided assays, performed by laboratories at the
manufacturing facilities and also independently as coded
samples by commercial testing centers.
Preparation of water extracts of I'm-Yunity™ (PSP)
To prepare water extracts of I'm-Yunity™ (PSP), contents
of each capsule (containing 340 mg powder) were suspended in 3.3 ml of water. The suspension was stirred
with intermittent mixing at 150 rpm for 60 minutes at
room temperature. The insoluble material was removed
by centrifugation in a micro-centrifuge. The soluble supernatant was sterilized by passing through a 0.22 µm filter
and kept in aliquots at 4°C. Water extracts of I'm-Yunity™
were analyzed by SDS-PAGE followed by staining with
Coomassie blue, also using the Schiff's reagent from
Sigma (this reagent is modified from the Periodic acidSchiff (PAS) method and is designed to identify glycoproteins), and with Rapid-Ag-Stain from MP Biomedicals
(Figure 1A–C). Staining with Coomassie blue revealed a
major and minor band migrating with average molecular
weights of 45-kDa and 35-kDa, respectively (Figure 1A).
Gels stained with the Schiff's reagent showed a magenta
pattern with a very light pink background; the most
intensely stain corresponded to a very broad protein band
migrating in the vicinity of >45-kDa (Figure 1B). Silver
staining did not produce a distinct pattern (Figure 1C). As
a whole, these results supported the interpretation that
water extracts of I'm-Yunity™ (PSP) contain highly heterogeneous polysaccharo-peptides, with variable carbohydrate moieties linked to different side chain
monosaccharides.
Cell culture and growth inhibition assay
Human HL-60 and U-937 leukemia cells were obtained
from the American Type Culture Collection (ATCC, Rockville, MD), and cultured as described [5,28,32-35]. For
treatment of either cell type, the stock aqueous extract of
I'm-Yunity™ (PSP) was first diluted in tissue culture media
and then added to cultured cells to give the final indicated
doses. In a typical experiment, 5 ml of cells at a density of
1 × 105 cells/ml were seeded in T25 flasks. Next, different
amounts of aqueous extracts of I'm-Yunity™ (PSP) were
added into the culture media. At the specified times, control and treated cells were harvested. Cell count was performed using a hemocytometer and cell viability was
determined by trypan blue exclusion [5,28,34,35]. In
addition, cell viability of control and treated cells was also
measured using the MTT assay [35]. Harvested cells were
washed twice with PBS, and pellets were stored at -80°C
for additional biochemical and molecular analyses.
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Effects of I'm-Yunity™ (PSP) on cell cycle progression
Cell cycle phase distribution was assayed by flow cytometry. Following a 3-day treatment of HL-60 or U-937 cells
with different amounts of water extract of I'm-Yunity™
(PSP) (0.1, 0.5, and 1.0 mg/ml), cells were washed with
PBS and stained with 1.0 µg/ml DAPI containing 100 mM
NaCl, 2 mM MgCl2 and 0.1% Triton X-100 (Sigma) at pH
6.8, as described [28,32,36-38]. The DNA-specific DAPI
fluorescence was excited with UV light emitting laser (NiCad), and collected with appropriate filters in an ICP-22
(Ortho Diagnostic, Westwood, MA) flow cytometer. MultiCycle software from Phoenix Flow Systems (San Diego,
CA) was used to deconvolute the cellular DNA content
histograms to obtain quantitation of the percentage of
cells in the respective phases (G1, S and G2/M) of the cell
cycle. Flow cytometry was also used to show cells undergoing apoptosis, evident by the appearance of the sub-G1
peak [32,39].
RNA isolation and quantitative real-time PCR of
cyclooxygenase 2 (COX2)
COX2 mRNA expression was assayed using semi-quantitative RT-PCR or quantitative real-time PCR [34,40-42].
Total cellular RNA was isolated from day 3 control and
I'm-Yunity™ (PSP) treated HL-60 cells using TRIzol reagent (InVitrogen) according to protocols provided by the
manufacturer. RNA purity and quantitation was determined by agarose gel electrophoresis and A260/280 absorbance ratio. First strand cDNA synthesis used 2 µl total RNA
incubated at 42°C for 50 min with Superscript RNase Hreverse transcriptase (InVitrogen). A 10-fold diluted
cDNA served as the template for PCR. To amply and quantitate the human COX2 gene, PCR was performed using
the Lightcycler PCR system (Roche Diagnostics, Mannheim, Germany) in glass capillaries in a final volume of
20 µl with addition of 1× Lightcycler master mix, 3 mM
MgCl2, 1 µM of COX2 primer sets (sense 5'-ATG GGG
TGA TGA GCA GTT GT-3' and antisense 5'-TGA GGC AGT
GTT GAT GAT TTG-3'), and 2 µl of cDNA template or
external standard template. The amplification reaction
consisted of heating samples at 95°C for 30 s followed by
40 cycles of heating at 20°C/s to 95°C with a 1-s hold,
cooling at 20°C/s to 55°C with a 1-s hold, and heating at
20°C/s to 72°C with a 10-s hold. A melting curve was
generated by heating the product at 20°C/s to 95°C, cooling it at 20°C/s to 60°C, and slowly heating at 0.2°C/s to
95°C, with fluorescence collection at 0.2°C intervals.
Human COX2 was amplified by conventional PCR and
cloned into PCR 2.1-TOPO vector (InVitrogen) for transformation in TOP10 competent cells. Plasmid DNA containing human COX2 was purified using the Mini Plasmid
Extraction Kit (Qiagen, Valencia, CA). Real-time PCR was
performed using the Light Cycler DNA Master SYBR Green
1 kit according to manufacturer's instructions. Ten fold
http://www.biomedcentral.com/1472-6882/6/30
serial dilutions of the purified plasmid DNA containing
100 to 107 copies of COX2 in 2 µl were used as standards
to calculate COX2 mRNA copy number, which is
expressed as % of control. The data obtained were analyzed using the Lightcycler software provided by the manufacturer. Only log-linear portion of amplification was
chosen for analysis. Experiments were performed in duplicate.
Preparation of whole cell extracts and western blot
analysis
Cells were suspended in buffer (50 µl/106 cells) containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1
mM PMSF, 5 µg/ml each of aprotinin, pepstatin, leupeptin, and lysed by 3 freeze/thaw cycles [33,42,43]. The
extracts were centrifuged and the clear supernatants were
stored in aliquots at -70°C. Ten to 20 µg of proteins were
separated on 10% SDS-PAGE. Membranes were probed
for expression of Rb, poly(ADP-ribose) polymerase
(PARP), bcl-2, survivin, bax, cytochrome c, p50 and p65
forms of NF-κB, STAT1, ERK, and β-actin. Commercial
antibodies (from Santa Cruz Biotechnology, Inc.) were
used at a dilution of 1:1000. Immunoreactivity was demonstrated by enhanced chemiluminescence (ECL) or color
reaction, using the manufacturer's protocol (Kirkegared &
Perry Laboratories).
Preparation of subcellular fractions from control and
treated HL-60 cells
Cytosolic and mitochondria fractions were obtained
using the Mitochondria isolation kit from Sigma. Harvested control and treated cells washed with ice-cold PBS
were suspended in extraction buffer containing 10 mM
Hepes, pH 7.5 supplemented with 200 mM mannitol, 70
mM sucrose, 1 mM EGTA, and 0.5 mg/ml BSA. The cell
suspension was homogenized by 30 passages through a
25-gauge needle. The homogenate was centrifuged at 600
× g, 5 min and the supernatant transferred to a fresh tube.
Further centrifugation of the supernatant at 11,000 × g, 10
min yielded a cytosolic supernatant and a mitochondria
pellet. The pellet was resuspended in extraction buffer and
centrifuged at 11,000 × g, 10 min. The mitochondria pellet was resuspended in 10 mM Hepes, pH 7.4 containing
250 mM sucrose, 1 mM ATP, 0.08 mM ADP, 5 mM
sodium succinate, 2 mM K2HPO4, and 1 mM dithiothreitol. Efficiency of separation of cytosolic and mitochondrial fractions was confirmed by gel electrophoresis and
immunoblotting using the mitochondria specific cytochrome c oxidase antibody (from Molecular Probes).
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U-937
B
120
Cell viability (%)
Cell growth (3 day)
% of control
A
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100
80
60
40
20
0
0
120
100
80
60
40
20
0
80
60
40
20
0
0
0.1 0.5 1.0 mg/ml
HL-60
D
Cell viability (%)
Cell growth (3 day)
% of control
C
0.1 0.5 1.0 mg/ml
0.1
0.5
1.0 mg/ml
HL-60
120
100
80
60
40
20
0
0
U-937
120
100
0
0.1 0.5 1.0 mg/ml
Control
Figure 2of cell growth and viability in U-937 and HL-60 cells by water extracts of I'm-Yunity™ (PSP)
Control of cell growth and viability in U-937 and HL-60 cells by water extracts of I'm-Yunity™ (PSP). Both cell types were cultured and treated with different dose of I'm-Yunity™ (PSP) as described in Materials and Methods. Panels A and C show suppression of proliferation of U-937 (panel A) and HL-60 (panel C) cells by increasing concentrations of I'm-Yunity™ (PSP).
Panels B and D show reduction of cell viability in U-937 (panel B) and HL-60 (panel D) by I'm-Yunity™ (PSP). Results in panels
A-D represent average ± SD of 3–4 experiments.
Results
Control of proliferation and cell cycle progression by I'mYunity™ (PSP) in HL-60 and U-937 cells
In previous studies, we demonstrated in HL-60 cells that
aqueous extracts of I'm-Yunity™ (PSP) exerted more
potent growth inhibitory and apoptosis-inducing effects,
compared to 70% ethanolic extracts [28]. Moreover, the
reduction in proliferation was accompanied by accumulation of cells in G1 simultaneous with a reduction in proportion of cells in the S phase [28]. To investigate whether
the growth inhibitory effects of aqueous extracts of I'mYunity™ (PSP) can be extended to other myeloid leukemia
cells, increasing concentrations of water extracts was
added to U-937 cells. A 72 h treatment resulted in significant inhibition of cell proliferation (Figure 2A). Time
course studies showed that suppression of growth was evident as early as 24 h after treatment (data not shown). Furthermore, suppression of proliferation was accompanied
by a decrease in cell viability using high but not low doses
(Figure 2B) of water extracts of I'm-Yunity™ (PSP), suggesting that the polysaccharo-peptide exerts complex anticellular effects in the U-937 cells. Similar to our previous
observations [28], treatment of HL-60 cells with the same
dose range of water extracts of I'm-Yunity™ (PSP) was
accompanied by a pronounced inhibition of cell growth
and viability (Figure 2C and 2D).
To explore further the manner by which extracts of I'mYunity™ (PSP) affected cell growth in these two cell types,
flow cytometric analysis was performed and the results
illustrated in Figure 3A. In U-937 cells, low dose (0.1 mg/
ml) induced an accumulation of cells in the S phase, concomitant with a decrease in proportion of cells in the G1
phase (Figure 3A). At higher concentrations (0.5 and 1.0
mg/ml), however, a reduction in S phase, together with an
increase in G2M phases of the cell cycle was observed.
These results provide further support for the notion that a
complex mechanism underlies the interaction between
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A
U-937
100%
Percentage of cells
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HL-60
90%
80%
Apoptosis
G2M
S
G1
70%
60%
50%
40%
30%
20%
10%
0%
0 mg/ml
Apoptosis
B
0
0.1
mg/ml
0.5
mg/ml
1 mg/ml
0.6
r0.3
12.1
r0.3
33.1
r0.3
0 mg/ml
0.1
mg/ml
0.5
mg/ml
1 mg/ml
42.8
r6.3
G2M
5.5
r0.6
5.6
r1.3
12.5
r1.8
20.5
r2.2
11.4
r1.0
9.0
r0.2
7.1
r0.4
14.9
r3.5
S
55.1
r2.8
69.4
r3.3
44.7
r3.2
37.9
r3.4
41.2
r1.1
37.2
r0.6
27.6
r1.3
11.3
r7.1
G1
39.4
r 2.5
25.0
r2.9
42.8
r3.9
41.5
r5.7
47.4
r0.1
53.9
r0.4
65.3
r1.7
73.8
r3.5
0.1
0.5
Day 1
0.1
0.5
1.0
0
Day 2Day 3
0.1
0.5 1.0
0
1.0
mg/ml
Rb
110-kDa
Actin
44-kDa
1
0.82
0.75 0.65
1
0.85
0.70.7
1
0.85 0.75 0.72
fold
Figure
mg/ml
Effects
tribution
I'm-Yunity™
of3changes
water extracts
and
(PSP)
induction
ofare
I'm-Yunity™
presented
of apoptosis
in
(PSP)
panel
inon
U-937
A
cellular
andDNA
HL-60content
cells treated
frequency
for 3histograms
days of treatment
showingwith
the cell
0, 0.1,
cycle
0.5phase
and 1.0
disEffects of water extracts of I'm-Yunity™ (PSP) on cellular DNA content frequency histograms showing the cell cycle phase distribution changes and induction of apoptosis in U-937 and HL-60 cells treated for 3 days of treatment with 0, 0.1, 0.5 and 1.0
mg/ml I'm-Yunity™ (PSP) are presented in panel A. Flow cytometric analysis was performed as described in Materials and
Methods and results shown represent average ± SD of 2 separate experiments. Panel B. Effects of I'm-Yunity™ (PSP) on Rb
expression in treated HL-60 cells. Relative changes in expression of Rb in control and days 1–3 treated cells were determined
by Western blot analysis as described in Materials and Methods. The intensity of the Rb and actin immunoreactive bands was
quantitated by densitometry and the actin-adjusted Rb expression levels are presented as fold differences, with the control
value for each day of treatment showing as 1.
extracts of I'm-Yunity™ (PSP) and U-937 cells. In HL-60
cells, however, a dose-dependent increase in G1, accompanied by a marked reduction in S phase of the cell cycle
resulted from treatment with water extracts of I'm-Yunity™
(PSP) (Figure 3A). These results raise the possibility that
water extracts of I'm-Yunity™ (PSP) target both cell cycle
checkpoints, as a likely contributing factor for its anti-proliferative activities in tumor cells. To gain further insights
on how water extracts of I'm-Yunity™ (PSP) might trigger
G1/S cell cycle arrest in HL-60 cells, we determined the
expression of the retinoblastoma gene product Rb, known
to play a pivotal role in controlling the G1/S phase transition via a cyclin kinase-mediated, switch on/off mechanism of the binding of transcription factor E2F [33,43,44].
Immunoblot analysis showed a dose-dependent inhibition of Rb expression in treated cells (Figure 3B). The
extent of reduction of Rb was correlated with the observed
cell cycle changes, suggesting that the attenuation of Rb
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A
Control (3 day)
B
1.0 mg/ml
Apoptosis
U-937
Cell Number
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HL-60
Apoptosis
U-937
0
0.5
0.1
0.5
bax
1
2.93
6.15
1
3.27
2.02
cytochrome c
Cytochromec
oxidase
25.6-kDa
HL-60
112-kDa
1
0.31
0.71
0.05
fold
89-kDa
1
0.27
0.77
3.37
fold
Cyto
0
0.5 0.75 mg/ml
23-kDa
bcl-2
26-kDa
mg/ml
cytochrome c
11-kDa
23-kDa
cytochrome c oxidase
0.74
0.59
survivin
1
actin
fold
0.81
0.46
0.5 0.75
0
25.6-kDa
44-kDa
actin
cytosol
fold
mitochondria
bax
1
16
24.3
1
1.5
3.4
fold
fold
bcl-2
1
2.4
0.8
1
0.7
0.7
fold
16.5-kDa
bax/bcl-2 ratio
1
6.7
30.4
1
2.1
4.9
cytochrome c
1
21.2
10.9
1
2.9
12.3
26-kDa
1
Mito
bax
11-kDa
bcl-2
mg/ml
112-kDa
89-kDa
DNA content
0
1
PARP
D
C
0.75
fold
fold
44-kDa
Figure HL-60
Changes
treated
4in cellcells
cycle phase distribution and apoptogenic/anti-apoptotic regulatory proteins in control and I'm-Yunity™ (PSP)Changes in cell cycle phase distribution and apoptogenic/anti-apoptotic regulatory proteins in control and I'm-Yunity™ (PSP)treated HL-60 cells. Panel A. Cellular DNA content frequency histograms showing the cell cycle phase distribution changes of
U-937 and HL-60 cells following 3 day treatment with 1.0 mg/ml doses of I'm-Yunity™ (PSP). Flow cytometric analysis was performed as described in Materials and Methods. Induction of apoptosis in treated cells is illustrated by cells showing fractional
DNA content. Panels B-C, changes in apoptogenic/anti-apoptotic proteins in control and 3 day I'm-Yunity™ (PSP)-treated HL60 cells. Expression of bax, cytochrome c, bcl-2, survivin, and PARP, identified by their respective molecular weights, was
determined by immunoblot analysis, then adjusted for protein loading using cytochrome c oxidase in panel B, or actin in panel
C, and presented as fold differences, with the control value showing as 1. Panel D, changes in bax, bcl-2 and cytochrome c in
the cytosolic and mitochondria fractions separated using the procedure detailed in Materials and Methods. Expression of the
various proteins was adjusted for loading using either cytochrome c oxidase (for mitochondria fraction) or actin (for cytosolic
fraction) and presented as fold differences, with the control value showing as 1. The adjusted bax and bcl-2 values were used to
calculate the ratio of bax/bcl-2 expression.
expression likely plays a critical role in restricting cell progression from G1 to S phase, in HL-60 cells.
Induction of apoptosis in I'm-Yunity™ (PSP)-treated HL60 cells
Treatment with high dose (1.0 mg/ml) of water extracts of
I'm-Yunity™ (PSP) for 3 days also resulted in the induction of apoptosis in both U-937 and HL-60 cells, as evi-
dent by the appearance of cells displaying fractional DNA
contents in flow cytometric analysis (Figure 4A), and further supported by the cleavage of DNA repair enzyme
PARP from its native 112-kDa form to an inactive 89-kDd
product (Figure 4B). Since disruption of the mitochondria, accompanied by changes in the relative subcellular
distribution of apoptogenic and anti-apoptotic regulatory
proteins, has been suggested as being among the key
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events that occur during apoptosis [37,45-50], we analyzed in detail changes in expression of apoptogenic and
anti-apoptotic proteins, notably, bax, cytochrome c, survivin, and bcl-2. Figure 4C shows composite changes in
these proteins in day 3, 0.1 and 0.5 mg/ml I'm-Yunity™
(PSP)-treated whole cell lysates. A pronounced increase in
bax and cytochrome c was clearly observed, which corresponded to significant decreases in levels of bcl-2 and survivin. As additional proof of the observed protein
changes, we analyzed bax, bcl-2 and cytochrome c in the
mitochondria and cytosolic fractions. Figure 4D shows
dose-dependent increases in the expression of bax in
mitochondria and correspondingly cytochrome c in the
cytosol; purity of separation into the respective subcellular compartments was supported by the distinctive presence of cytochrome c oxidase in the mitochondria and its
absence in the cytosolic fraction. Induction of apoptosis is
further demonstrated by dose-dependent I'm-Yunity™
(PSP)-elicited increase in ratio of bax/bcl-2 (Figure 4D).
Dose-dependent increase of bax and correspondingly,
reduction in bcl-2 expression can be detected as early as
30–36 hours after treatment (data not shown).
Down-regulation of NF-κB by water extracts of I'mYunity™ (PSP)
NF-κB is a transcription factor that has been implicated in
the control of cell growth [51-53]. Constitutive activation
of the NF-κB family of transcription factors in various
human tumors has been hypothesized to contribute to the
development and/or progression of malignancy by regulating the expression of gene sets involved in cell growth
and proliferation, circumvention of apoptosis, and the
induction of angiogenesis and metastasis [52,53]. We
therefore measured changes in the steady state level of NFκB in HL-60 cells treated with water extracts of I'mYunity™ (PSP). Pronounced decreases in p65 form and to
a lesser degree also p50 form of NF-κB was found in I'mYunity™ (PSP)-treated HL-60 cells in day 3, varying doses
of I'm-Yunity™ (PSP)-treated cells (Figure 5A). Figure 5B
showed the quantitive determination of changes in both
p65 and p50 forms of NF-κB using a single dose of 0.5
mg/ml water extracts of I'm-Yunity™ (PSP).
NF-κB is involved in transcriptional control of cyclooxygenase 2 (COX2) [54-57]. Therefore, to obtain information on functional consequences associated with the
suppression of NF-κB by I'm-Yunity™ (PSP), we determined COX2 expression using quantitative real-time and
semi-quantitative RT-PCR. Results in Figure 6 showed that
HL-60 cells treated with water extracts of I'm-Yunity™
(PSP) were accompanied by a 65–85% reduction in COX2
expression, depending on the assays used.
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Modulation of extracellular signaling protein expression
by I'm-Yunity™ (PSP)
Survival of cells in multicellular organisms is inextricably
linked to and dependent on cues from extracellular
growth factors. Availability of growth factors and signaling events they elicit contribute to the homeostatic balance between cell proliferation and death. In tumor cells,
their endowment with oncogenes together with the hyperactive status of oncogene products often result in amplified and/or persistently activated growth and survival
pathways.
Cultured tumor cells incubated with extracts of I'mYunity™ (PSP) are accompanied by significant changes in
the expression of several important growth modulators
and cytokines [28]. Similarly, patients treated with I'mYunity™ (PSP) showed increases in interferon-γ expression, a cytokine known to have profound immune regulatory properties [25]. Conceivably, interferon-γ could
contribute to the suppression of tumorigenesis indirectly,
through modulation of the immune system, as well as
directly by exerting anti-proliferative and pro-apoptotic
effects. Both mechanisms, in principle, may invoke regulation of gene expression mediated through the STAT (signal transducer and activator of transcription) proteins
[58-62]. Recent studies have shown, for example, that
STAT1, as a member of the STAT protein family, plays a
vital role in the control of apoptosis; namely, lymphocytes from STAT1-/- mice failed to undergo apoptosis,
had reduced cytokine processing and apoptogenic proteins caspase 1 and 11, and demonstrated increased proliferation in vitro, compared to cells from wild-type
animals [63].
It is therefore of interest to determine whether treatment
of HL-60 cells with water extract of I'm-Yunity™ (PSP)
may lead to changes in STAT1. Immunoblot analysis
showed a dose-dependent STAT1 increase in day 3, I'mYunity™ (PSP)-treated cells (Figure 7). In contrast, analysis
of another prominent survival pathway, mediated by the
extracellular signal-regulated kinase (ERK), showed that
treatment with I'm-Yunity™ (PSP) reduced levels of activated, phosphorylated ERK, while total ERK was
unchanged, compared to control cells (Figure 7).
Discussion
A significant percentage of the estimated two million
deaths recorded in the United States each year are attributable to cancer and its ensuing complications. Cancer
presents clinically as a constellation of diseases and at all
stages – from primary prevention to treatment – can be
profoundly and significantly affected by nutrition and
diets. The existence of such a link is well supported by decades of epidemiological studies showing that the consumption of plant-based diets is directly associated with
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A
B
Day 3
0.1
control
0.3
0.5
mg/ml
NF-NB (p65)
65-kDa
NF-NB (p50)
50-kDa
Actin
44-kDa
Protein expression
(Arbitrary unit)
0
100
80
NF-NB ( p 6 5 )
0.5 mg/ml
60
40
20
1
2
Time (day)
120
3
100
50
80
Protein expression
(Arbitrary unit)
Protein expression (% of control)
0
60
40
20
0
0 0.1 0.3 0.5
0 0.1 0.3 0.5 mg/ml
NF-NB(p65)
NF- NB(p50)
NF-NB ( p 5 0 )
control
40
0.5 mg/ml
30
20
10
0
1
2
Time (day)
3
Regulation
Figure 5 of expression of NF-κB in HL-60 cells by water extracts of I'm-Yunity™ (PSP)
Regulation of expression of NF-κB in HL-60 cells by water extracts of I'm-Yunity™ (PSP). Control and 3 day I'm-Yunity™
(PSP) treated cells were harvested and total protein extracts were prepared, separated by SDS-PAGE and analyzed for expression of NF-κB p65, p50 and actin by immunoblot analysis (panel A). Time-dependent changes in actin-adjusted, NF-κB expression by water extracts of I'm-Yunity™ (PSP) are shown in panel B.
reduced risk of cancer [64-76]. The impact of nutrition
and diets in prevention of carcinogenesis is formally conceptualized in chemoprevention by Sporn and others in
the 1970s, which emphasizes the use of pharmacologic or
natural agents to inhibit development or progression of
invasive cancer [67-69,71,72,74,77-79]. Research in
recent years has begun to focus on "bioactive compounds" in diets with chemopreventive attributes and
delineation of their mechanism(s) [67,70,72].
Efficacy of chemopreventive agents may have multiple
mechanistic bases: reversal of abnormal differentiation,
suppression of cell replication/growth, and induction of
apoptosis. Additionally, inhibition of carcinogen activation and increase in their removal by detoxification
enzymes are also considered prudent cancer prevention
strategies [71,72,74]. Since many of the very same chem-
opreventive attributes are found in I'm-Yunity™ (PSP), we
propose that it, as an example of mushroom and mushroom products, may be included in this category of dietary agents. Indeed, based results from this
communication and earlier studies, it may be suggested
that I'm-Yunity™ (PSP) is an easily compliant adjunctive
treatment particularly germane to human leukemia and
other blood-borne hematologic malignancies. Since
polysaccharide moieties predominate in I'm-Yunity™
(PSP), it is possible that less chemo- and radio-resistant
clones will result from treatment using such an agent.
The cellular changes reported in this communication as a
result of treatment with water extracts of I'm-Yunity™
(PSP) can be explained by the multiple effects it elicits,
which include suppression of the retinoblastoma tumor
suppressor protein Rb, increased release of cytochrome c
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COX2 expression
(% of control)
A
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Day 3
120
0
100
0.1
0.5
1.0
mg/ml
44-kDa
fold
ERK-P
80
60
1
0.65
0.70
0.49
1
0.95
1.00
1.00fold
1
1.66
7.18
8.14
ERK
40
20
42/44-kDa
STAT1
0
0 mg/ml0.1 mg/ml0.5mg/ml
Actin
110-kDa
fold
44-kDa
B
0
0.1
0.5
mg/ml
COX2 (724 bp)
GAPDH (598 bp)
1
0.16
0.35
(fold)
Figure 6of I'm-Yunity™
Suppressed
extracts
expression of(PSP)
COX2 in HL-60 cells by water
Suppressed expression of COX2 in HL-60 cells by water
extracts of I'm-Yunity™ (PSP). Control and treated cells
were harvested and expression of COX2 was determined
quantitatively by real-time PCR (panel A) and semi-quantitatively by RT-PCR (panel B). Semi-quantitative expression of
COX2 in panel B was also adjusted using expression of
GAPDH and presented as fold differences, with 1 representing the value in control cells.
from mitochondria to the cytosol, reduced expression of
anti-apoptotic protein bcl-2 and survivin, and simultaneously, increase in bax, as well as a disruption in cell signaling events, as collectively depicted in the scheme in
Figure 8.
A number of new molecular targets and mechanistic
insights have surfaced from the present studies. First and
foremost is the observation that I'm-Yunity™ (PSP) downregulates the expression of NF-κB in HL-60 cells, thereby
identifying a new, novel molecular target of this family of
bioactive polysaccharo-peptides. It is noteworthy that the
effect of I'm-Yunity™ (PSP) on NF-κB correlated well with
the suppression of growth, suggesting a causal relationship. NF-κB is a transcription factor known to be functionally associated with cell survival; its constitutive activation
in various human malignancies contributes to tumor
transformation, development, and progression. These
effects may relate to the ability of NF-κB to control the
transcription of genes involved in cell growth and proliferation, suppression or escape of apoptosis, acquisition of
new vasculature and blood supply, and dissemination to
Figure 7changes
Relative
(PSP)-treated
HL-60
in STAT1
cells and ERK levels in I'm-Yunity™
Relative changes in STAT1 and ERK levels in I'm-Yunity™
(PSP)-treated HL-60 cells. Control and treated cells were
harvested and total protein extracts were prepared, separated by SDS-PAGE and analyzed for expression of STAT1
and ERK by western blot analysis, with actin expression used
to adjust for protein loading.
distant location for further growth and colony formation
[52-56]. Treatment of HL-60 cells with water extract of
I'm-Yunity™ (PSP) caused a precipitous reduction in p65
and to a lesser degree also p50 subunits of NF-κB (Figure
5). This finding is significant since it proffers the notion
that this family of polysaccharo-peptides acts in part by
forestalling a pivotal factor required for the survival and
propagation of tumor cells. The more pronounced decline
in p65 relative to p50 may relate to the fact that these two
subunits of NF-κB are known to be different gene products and not necessarily regulated in a coordinate fashion.
Studies have shown, for example, that p50 is proteolytically derived from a p105 precursor and hence, in principle, could be subjected to more complex mechanism of
control, compared to the p65 subunit. Taking these into
consideration, it is entirely possible that the p65 and p50
subunits are differentially regulated depending on the
dose of I'm-Yunity™ (PSP) used for treating the cells. Conceivably, therefore, low rather than high doses of I'mYunity™ (PSP) are effective in suppressing the expression
of the p50 subunits. In contrast, a broader dose range of
I'm-Yunity™ (PSP) affects the expression of the p65 subunit. It should also be pointed out that our findings do not
necessarily contradict the role of I'm-Yunity™ (PSP) as an
immune modulator, and similarly, other data pointing to
NF-κB as a tumor suppressor rather than a promoter of
tumorigenesis [52,54]. It may well be that in normal
immune cells, I'm-Yunity™ (PSP) at an appropriate dose
range does indeed increases the expression of NF-κB and
thereby promotes the increased transcription of genes in
immune cells conducive to a heightened immune status.
The same cell type consideration may equally apply to
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I’mI’m-YunityTM (PSP)
Cell Membrane
I’mI’m-YunityTM (PSP)
Disruption of cell signaling
ERK1/2p
NF-NB
Rb p
G1/Sp
??
??
Anti-apoptotic
ƒ bcl-2 p
ƒ survivin p
COX2p
Growthp
STAT1 n
Pro-apoptotic
ƒ bax n
ƒ cytochromec n
ƒ PARP cleavage n
Apoptosisn
Figure 8 mechanism of action of I'm-Yunity™ (PSP)
Proposed
Proposed mechanism of action of I'm-Yunity™ (PSP). In this model, the ability of water extracts of I'm-Yunity™ (PSP) to inhibit
cell proliferation and induce apoptosis in cancer cells is hypothesized to involve multiple effects, including 1) disruption of cell
cycle control machinery, 2) perturbation in apoptogenic/anti-apoptotic regulatory protein expression promoting the induction
of apoptosis, 3) alteration in mitogenic signaling pathways, 4) regulation of gene expression, exemplified by suppression of cell
survival transcription factor NF-κB.
recent evidence showing that NF-κB can function as a
tumor suppressor by actively repressing anti-apoptotic
genes. The acquisition of a non-conventional role for NFκB, i.e., functioning in tumor suppression rather than promotion, may also depend on stimuli impinging on cells,
and just as plausible, the co-expression of genes acting in
concert with NF-κB to facilitate transcriptional repressor
of anti-apoptotic genes. It has been demonstrated, for
example, that ARF (ADP-ribosylation factor) and p53 as
well as certain inducers of NF-κB DNA-binding activity,
such as ultraviolet light and some chemotherapeutic compounds, can induce the association of NF-κB subunits
with transcriptional corepressor complexes, allowing
them to function as repressors rather than activators of
gene expression [80-82]. Whether these considerations
can apply to HL-60 cells, known to have p53-null status
but do express the ARF [83-89], in the context of cellular
effects elicited by I'm-Yunity™ (PSP) remain to be studied.
Further research will also focus on investigation of
detailed changes in steady state levels and subcellular distribution of NF-κB, using control and treated cells harboring a stably transfected dominant-negative IκB gene in
which amino acid serine at positions 32 and 34 of IκB is
mutated to amino acid alanine. These changes should
exclude its phosphorylation by IκB kinase, which is
required for the degradation of IκB, coincident with the
cytoplasm-to-nucleus translocation of NF-κB [51,90,91].
A second new finding revealed in the present studies is the
ability of I'm-Yunity™ (PSP) to down regulate COX2
expression. Persistent chronic inflammation plays an
important role in carcinogenesis [92-94]. In the early
phase of inflammation, COX2 facilitates the production
of inflammatory prostaglandins. COX2 has been shown
to be overexpressed in a variety of cancers [95-97]. Further, there is compelling evidence from in vitro experimental studies that inhibition of COX2 decreases cellular
proliferation, increases apoptosis, and modulates genes
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involved in cell cycle regulation [93,94]. Therefore we
measured COX2 expression in control and treated cells.
Results in Figure 6 show that COX2 expression was significantly suppressed by treatment with water extracts of I'mYunity™ (PSP).
charged mucopolysaccharide) [102-104] or heparan sulfate, e.g., chondroitin sulfate [105-107]. Notably, some of
these possibilities can be tested experimentally and are
being considered for further investigation in our laboratory.
A third important observation relates to the modulation
of potent signaling molecules by I'm-Yunity™ (PSP), as
exemplified by an increase the expression of STAT1 in parallel with the suppression of the phosphorylated form of
ERK (Figure 7). STATs and their protein partners JAKs
(Janus kinases) are evolutionarily conserved signaling and
cellular communication molecules that enable cytokines
to mount a repertoire of host defenses and immunological responses. Similarly, ability of cells to respond to extracellular challenges also depend upon signaling pathways
governed by the MAP kinase modules, with sequentially
acting protein kinases including ERK as the archetypal
form. In both examples, interaction of ligand or an external challenge with membrane-associated receptors are
considered to be key initiation events. Because of the
importance of the STAT and ERK signaling pathways in
homeostatic control of cells, as discussed, it is of interest
and pertinent to ask how they might be affected by I'mYunity™ (PSP). One possibility is that I'm-Yunity™ (PSP),
having structural similarity to proteoglycans [4], act
directly as agonists/antagonists of specific surface receptor
ligands, and in turn, augment the trigger of the JAK/STAT
signaling pathway while concurrently dampen the events
directed to the ERK signaling cascade. A second possibility
is that I'm-Yunity™ (PSP) functions as a growth factor/
cytokine decoy to sequester/trap the relevant growth factor/cytokine required for HL-60 cellular function, or bind
the pertinent receptor in ways that block further engagement and interaction with their physiological, cognate ligands. This possibility may be supported by structural
resemblance existing between the bioactive constituents
present in I'm-Yunity™ (PSP) with proteoglycans occurring in the extracellular matrix of cancer cells [98]. As
mentioned in the Introduction, a broad spectrum of bioactive molecules, ranging from those with defined structures and functions, e.g., proteases, nucleases, to others
with heterogeneous structures and ill-defined activities,
e.g., polysaccharo-peptides and polysaccharides, have
been identified and isolated from mushrooms
[2,4,99,100]. Finally, it is possible that I'm-Yunity™ (PSP)
exerts its effect on STAT1 and ERK by disrupting intracellular signaling events, secondary to its internalization that
may involve the formation of a complex between the
polysaccharo-peptides with specific components in the
serum to facilitate its uptake into cells. This mechanism
may draw analogy from lipid-mediated delivery of highly
charged and heterogeneous plasmid DNA into cultured
mammalian cells and as vaccines [101], as well as the
intestinal absorption of oral heparin (a heterogeneous,
Conclusion
Treatment of human leukemia cells by water extracts of
I'm-Yunity™ (PSP) causes cell cycle arrest and alterations
in the expression of apoptogenic/anti-apoptotic and
extracellular signaling regulatory proteins, the net result
being a reduction in proliferation and an increase in
apoptosis. These findings may be part of the mechanisms
that underlie or contribute to its reported clinical
attributes and overall health benefits.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TCH carried out tissue culture and flow cytometric studies
using the core facilities at the Brander Cancer Institute of
New York Medical College. TCH designed and performed
the real-time PCR analysis with assistance from a former
post-doctoral fellow Xiaohua Lu. PW isolated and purified
cytosol from mitochondria and performed immunoblot
analysis of the isolated fractions in the revision of the
paper. SP performed immunoblot analysis using whole
cell lysates in the revision of the paper. TCH and JMW
designed the experiments and wrote the paper. All authors
read and approved the final manuscript.
Acknowledgements
Research in this report was supported in part by an unrestricted grant from
the Hong Kong Healthcare Center Ltd. We acknowledge with thanks the
unwavering support Ms. Vivien Chou and her family has provided for mechanistic studies of Chinese herbals in preventing and treating chronic diseases.
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